It is of course elementary that all microbial cells produce energy for their cellular activity through respiration. As cellular respiration occurs in living cells, pyridine nucleotides are reduced, flavins are oxidized and other coenzymes and metabolites are produced. Alternatively, spores are found to be abundant with a calcium dipicolinic acid complex (a fluorescent compound otherwise rare in nature). The oxidation state of pyridine nucleotides, flavins and other cofactors, and/or the presence of calcium dipicolinate, can be simultaneously elucidated by concurrent excitation of each component with the appropriate electromagnetic radiation followed by detection of the characteristic radiation emitted by these individual fluorophores. Simultaneous frequency-modulated excitation of a sample with multiple energies characteristic of the excitation for fluorescent cellular and endospore components with the subsequent collection and detection of emitted, reflected and scattered light energies (associated with the fluorophores, respectively) is fundamental for the detection of microbes in a sample or on a non-living surface by the method described herein. It will be appreciated by those skilled in the art that the use of frequency-modulated ultraviolet light excites aromatic amino acids and nucleic acids, some of whose emission is self-absorbed by the sample sequentially exciting calcium dipicolinate and pyridine nucleotides, some of whose emission is self-absorbed by the sample in turn exciting cofactors (e.g., flavins), part of whose emission is used to excite porphyrins and other flavins. The fluorescent emissions of the sample are collected and analyzed as described previously.
The detection of respiring cells in real world samples is made more reliable by the aforementioned method for two reasons. First, the simultaneous excitation of microbes by multiple excitation energies and ensuing coincident detection of numerous fluorescence signals reduces the chance of interference, as the probability of an interference source duplicating the characteristics of numerous fluorophores is extremely small. Second, the relative quantities of the intrinsic metabolites, and thus of the resulting fluorescent signals, have been found to fall within defined physiological ranges. This is true also of the sum of the resulting fluorescence signals associated with each excitation wavelength. Analysis of the signals is achieved with a method capable of two things: (1) separating the detected fluorescent signals originating from any microbes present from interferences or background signals and/or scattered excitation signals, and (2) a requirement that the sum of the intensities of the signals from microbial metabolites associated with each excitation frequency for microbial components and spore components fall within physiological ranges. Thus, the basis for the detection of microbes in a sample is comprised of the following steps: first, excitation of a sample simultaneously with multiple frequency-modulated excitation energies characteristic of cellular metabolites, microbial components, and spore components; second, the subsequent collection of the resulting frequency-modulated fluorescence signals; and finally, analysis of the collected signals with a method capable of removing background fluorescence and comparing the relative signal magnitudes of metabolites to known physiological ranges. In the present invention, since each excitation source is frequency modulated the resulting sum of fluorescence signals resulting from that excitation energy is similarly frequency modulated (for frequencies longer than the fluorescence lifetime of the excited intrinsic microbial fluorophore) and can be thus distinguished from any background signals by use of any analysis method capable of detecting only the resultant emission signals occurring at the frequency of the excitation radiation. One example of such an analysis method well known to those skilled in the art would be the use of the discrete Fourier transform on the detected fluorescence signals over a period of time that meets or exceeds the requirements of the Nyquist-Shannon sampling theorem.
Long-established technologies and methods used for microbial detection rely upon detection of products resulting from metabolic reactions, immunological capture or the amplification of expected nucleotide sequences. Since this invention employs detection of multiple intrinsic fluorophores from microbes, coupled with an analysis of the relative amount of signals due to these fluorophores, it can not only determine the presence of microbes, but is also capable of differentiating between viable cells, non-viable cells and spores. This method and apparatus uses no reagents, requires no physical contact with the sample, and delivers ‘real-time’ results.
There are other microbial detection methods that utilize fluorescence. Many of the flow cytometry methodologies rely on the fluorescence of dye molecules conjugated to immunological proteins targeted to the analyte of interest. An example of this can be found in U.S. Pat. No. 4,745,285 (to Recktenwald, et al.). Other fluorescence methods use added fluorescent metabolic dyes or dye conjugates (as in U.S. Pat. No. 4,900,934 to Peeters, et al.).
Some of the fluorescence-based microbe detection methodologies utilize intrinsic cellular fluorophores. One method (U.S. Pat. No. 5,424,959 to Reyes, et al.) simply compares the fluorescence spectra of the sample with a library of spectra. The method described in U.S. Pat. No. 5,474,910 to Alfano, compares the fluorescence of a sample surface to that of a clean surface. A popular intrinsic fluorophore used in microbial detection methods is the reduced pyridine nucleotide NADH. In U.S. Pat. No. 5,701,012 to Ho, NADH fluorescence is detected in a forced airstream containing the sample and compared to a blank. Alternatively, the ratio of NADH fluorescence to either the scattered excitation signal or other fluorescence emissions is used in U.S. patents to Powers (U.S. Pat. Nos. 5,760,406 and 5,968,766).
In U.S. Pat. Nos. 5,760,406 and 5,968,766, which issued 02 Jun. 1998 and 19 Oct. 1999, respectively, and which are incorporated herein by reference, there is disclosed a method and apparatus for the detection of microbes on non-living surfaces and samples. The sample to be examined is excited with electromagnetic radiation (1) having a wavelength greater than 350 nm causing the excitation of pyridine nucleotides present in microbial cells, and (2) having a wavelength below 340 nm as a measure of other characteristics of the environment. The ratios of the microbial pyridine nucleotide fluorescence emission (resultant from the excitation at the different wavelengths) to the reflected excitation signals are calculated and compared, as the basis for both the detection and quantitation of microbes present on the sample. This invention is able to locate and quantitate microbes on non-living surfaces, including meats.
Whereas the aforementioned patents to Powers depend upon ratio fluorescence for the detection of a single metabolite, the present invention utilizes excitation of one or more fluorophores coupled with an algorithm that subtracts the detected signals due to the scattered/reflected excitation energies. This difference in design and methodology makes the current invention able to detect and quantitate microbes on non-living surfaces, in liquids and in air relative to other fluorescence methods using only one detector that collects all the resulting sample fluorescence over time provided that excitation energies can be effectively optically filtered and thus prevented from reaching the detector.
In U.S. Pat. No. 6,750,006, which issued Jun. 15, 2004, multiple intrinsic fluorescence emission signals resultant from multi-wavelength excitation are used coupled with an algorithm that subtracts the contributions from reflected and scattered excitation energies. The current invention is differs from U.S. Pat. No. 6,750,006 in that the contribution from the background fluorescence is accounted for by the analysis method (like the Fourier transform of the time-dependant signal) and that it can utilize many fewer photodetectors. The present invention shares with U.S. Pat. No. 6,750,006 the benefit of the certain detection of microbes from the detection of multiple intrinsic fluorophores, reducing the probability of false positive results due to background interferences. The detection of microbes with the foregoing method and apparatus will have uses in biowarfare agent detection, cell sorting, medical diagnostics, sterilization verification, water quality testing, food production and preparation safety, and emergency response teams tasked with the detection, decontamination and protection of public infrastructure facilities.
With near constant announcements of bacterial contamination in foodstuffs (meats, poultry, seafood, juices, fruits and vegetables), there has been a need to provide a method and apparatus that can be used to detect such microbial contamination in foods, on foods and on food preparation surfaces. This method and apparatus, as an object of the invention, should be operated inexpensively and rapidly in, for example, meat and poultry production facilities.
It is yet another object of the invention to provide a method and apparatus for use in the detection of microbial contamination on foods in which the fluorescence of pyridine nucleotides, flavins and other cofactors and spore components are excited by electromagnetic radiation to distinguish the metabolic reactions and spore components of microbes from the tissue of foodstuffs, allowing microbial contamination on foods to be determined without contact with said food.
It is accordingly an object of the invention to provide a method and apparatus that can be used in the detection of microbial contamination on non-living surfaces, in liquids and air. As a specific object of the invention, the method and apparatus can be used to find microbes and microbial contamination inexpensively and rapidly in, for example, health-care facilities, research laboratories, water treatment and testing stations, public buildings and on the battlefield.
It is yet another object of the invention to provide a method and apparatus for use in the detection of microbial contamination on non-living surfaces and in liquid and air samples in which the fluorescence of pyridine nucleotides, flavins and other cofactors and spore components are excited by electromagnetic radiation to distinguish the metabolic reactions of microbes and/or presence of spores from the background of the media or scattering, allowing microbial contamination in samples to be determined without contact with said sample.
It is yet another object of the invention to provide a method and apparatus for use in the differentiation between viable cells, non-viable cells, spores and non-contaminated samples in which the fluorescence of pyridine nucleotides, flavins and other cofactors and spore components are excited by electromagnetic radiation with the differences in the relative quantities of the intrinsic fluorophores in each used to distinguish the presence of microbes from the background of the media or scattering.